Motor systems must continuously adapt their output to maintain a desired trajectory. While the spinal circuits underlying rhythmic locomotion are well described, little is known about how the network modulates its output strength. A major challenge has been the difficulty of recording from spinal neurons during behavior. Here, we use voltage imaging to map the membrane potential of large populations of glutamatergic neurons throughout the spinal cord of the larval zebrafish during fictive swimming in a virtual environment. We characterized a previously undescribed subpopulation of tonic-spiking ventral V3 neurons whose spike rate correlated with swimming strength and bout length. Optogenetic activation of V3 neurons led to stronger swimming and longer bouts but did not affect tail beat frequency. Genetic ablation of V3 neurons led to reduced locomotor adaptation. The power of voltage imaging allowed us to identify V3 neurons as a critical driver of locomotor adaptation in zebrafish.

Motor systems must continuously adapt their output to maintain a desired trajectory. While the spinal circuits underlying rhythmic locomotion are well described, little is known about how the network modulates its output strength. A major challenge has been the difficulty of recording from spinal neurons during behavior. Here, we use voltage imaging to map the membrane potential of large populations of glutamatergic neurons throughout the spinal cord of the larval zebrafish during fictive swimming in a virtual environment. We characterized a previously undescribed subpopulation of tonic-spiking ventral V3 neurons whose spike rate correlated with swimming strength and bout length. Optogenetic activation of V3 neurons led to stronger swimming and longer bouts but did not affect tail beat frequency. Genetic ablation of V3 neurons led to reduced locomotor adaptation. The power of voltage imaging allowed us to identify V3 neurons as a critical driver of locomotor adaptation in zebrafish.

As animals adapt to new situations, neuromodulation is a potent way to alter behavior, yet mechanisms by which neuromodulatory nuclei compute during behavior are underexplored. The serotonergic raphe supports motor learning in larval zebrafish by visually detecting distance traveled during swims, encoding action effectiveness, and modulating motor vigor. We found that swimming opens a gate for visual input to cause spiking in serotonergic neurons, enabling encoding of action outcomes and filtering out learning-irrelevant visual signals. Using light-sheet microscopy, voltage sensors, and neurotransmitter/modulator sensors, we tracked millisecond-timescale neuronal input-output computations during behavior. Swim commands initially inhibited serotonergic neurons via GABA, closing the gate to spiking. Immediately after, the gate briefly opened: voltage increased consistent with post-inhibitory rebound, allowing swim-induced visual motion to evoke firing through glutamate, triggering serotonin secretion and modulating motor vigor. Ablating GABAergic neurons impaired raphe coding and motor learning. Thus, serotonergic neuromodulation arises from action-outcome coincidence detection within the raphe, suggesting the existence of similarly fast and precise circuit computations across neuromodulatory nuclei.

Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid-protein interactions in general and for the mechanism of voltage sensing in particular.

1. The voltage- and space-clamp errors associated with the use of a somatic electrode to measure current from dendritic synapses are evaluated using both equivalent-cylinder and morphologically realistic models of neuronal dendritic trees. 2. As a first step toward understanding the properties of synaptic current distortion under voltage-clamp conditions, the attenuation of step and sinusoidal voltage changes are evaluated in equivalent cylinder models. Demonstration of the frequency-dependent attenuation of voltage in the cable is then used as a framework for understanding the distortion of synaptic currents generated at sites remote from the somatic recording electrode and measured in the voltage-clamp recording configuration. 3. Increases in specific membrane resistivity (Rm) are shown to reduce steady-state voltage attenuation, while producing only minimal reduction in attenuation of transient voltage changes. Experimental manipulations that increase Rm therefore improve the accuracy of estimates of reversal potential for electrotonically remote synapses, but do not significantly reduce the attenuation of peak current. In addition, increases in Rm have the effect of slowing the kinetics of poorly clamped synaptic currents. 4. The effects of the magnitude of the synaptic conductance and its kinetics on the measured synaptic currents are also examined and discussed. The error in estimating parameters from measured synaptic currents is greatest for synapses with fast kinetics and large conductances. 5. A morphologically realistic model of a CA3 pyramidal neuron is used to demonstrate the generality of the conclusions derived from equivalent cylinder models. The realistic model is also used to fit synaptic currents generated by stimulation of mossy fiber (MF) and commissural/associational (C/A) inputs to CA3 neurons and to estimate the amount of distortion of these measured currents. 6. Anatomic data from the CA3 pyramidal neuron model are used to construct a simplified two-cylinder CA3 model. This model is used to estimate the electrotonic distances of MF synapses (which are located proximal to the soma) and perforant path (PP) synapses (which are located at the distal ends of the apical dendrites) and the distortion of synaptic current parameters measured for these synapses. 7. Results from the equivalent-cylinder models, the morphological CA3 model, and the simplified CA3 model all indicate that the amount of distortion of synaptic currents increases steeply as a function of distance from the soma. MF synapses close to the soma are likely to be subject only to small space-clamp errors, whereas MF synapses farther from the soma are likely to be substantially attenuated.(ABSTRACT TRUNCATED AT 400 WORDS)

In rats, navigating through an environment requires continuous information about objects near the head. Sensory information such as object location and surface texture are encoded by spike firing patterns of single neurons within rat barrel cortex. Although there are many studies using single-unit electrophysiology, much less is known regarding the spatiotemporal pattern of activity of populations of neurons in barrel cortex in response to whisker stimulation. To examine cortical response at the population level, we used voltage-sensitive dye (VSD) imaging to examine ensemble spatiotemporal dynamics of barrel cortex in response to stimulation of single or two adjacent whiskers in urethane-anesthetized rats. Single whisker stimulation produced a poststimulus fluorescence response peak within 12-16 ms in the barrel corresponding to the stimulated whisker (principal whisker). This fluorescence subsequently propagated throughout the barrel field, spreading anisotropically preferentially along a barrel row. After paired whisker stimulation, the VSD signal showed sublinear summation (less than the sum of 2 single whisker stimulations), consistent with previous electrophysiological and imaging studies. Surprisingly, we observed a spatial shift in the center of activation occurring over a 10- to 20-ms period with shift magnitudes of 1-2 barrels. This shift occurred predominantly in the posteromedial direction within the barrel field. Our data thus reveal previously unreported spatiotemporal patterns of barrel cortex activation. We suggest that this nontopographical shift is consistent with known functional and anatomic asymmetries in barrel cortex and that it may provide an important insight for understanding barrel field activation during whisking behavior.

The last decade has seen a rapid increase in the number of tools to acquire volume electron microscopy (EM) data. Several new scanning EM (SEM) imaging methods have emerged, and classical transmission EM (TEM) methods are being scaled up and automated. Here we summarize the new methods for acquiring large EM volumes, and discuss the tradeoffs in terms of resolution, acquisition speed, and reliability. We then assess each method’s applicability to the problem of reconstructing anatomical connectivity between neurons, considering both the current capabilities and future prospects of the method. Finally, we argue that neuronal ’wiring diagrams’ are likely necessary, but not sufficient, to understand the operation of most neuronal circuits: volume EM imaging will likely find its best application in combination with other methods in neuroscience, such as molecular biology, optogenetics, and physiology.

Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging.

A system for a laser-scanning microscope includes an optical element configured to transmit light in a first direction onto a first beam path and to reflect light in a second direction to a second beam path that is different from the first beam path; a reflector on the first beam path; and a lens including a variable focal length, the lens positioned on the first beam path. The lens and reflector are positioned relative to each other to cause light transmitted by the optical element to pass through the lens a plurality of times and in a different direction each time. In some implementations, the system also can include a feedback system that receives a signal that represents an amount of focusing of the lens, and changes the focal length of the lens based on the received signal.

Mechanisms that entrain and drive rhythmic epileptiform discharges remain debated. Traditionally, this quest has been focusing on interneuronal networks driven by GABAergic connections that activate synaptic or extrasynaptic receptors. However, synchronised interneuronal discharges could also trigger a transient elevation of extracellular GABA across the tissue volume, thus raising tonic GABAA receptor conductance (Gtonic) in multiple cells. Here, we use patch-clamp GABA ‘sniffer’ and optical GABA sensor to show that periodic epileptiform discharges are preceded by region-wide, rising waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to mechanistic principles underpinning this relationship. We validate this hypothesis using simultaneous patch-clamp recordings from multiple nerve cells, selective optogenetic stimulation of fast-spiking interneurons. Critically, we manipulate GABA uptake to suppress extracellular GABA waves but not synaptic GABAergic transmission, which shows a clear effect on rhythm generation. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA actions in pacing regenerative rhythmic activity in brain networks.